Roles And Mechanisms Of Ghrelin In The Onset Of Female Precocious Puberty

Background:The initiation of puberty is mainly regulated by genetic factors,but environmental,nutritional,and epigenetic factors also play important roles in the onset of puberty.The morbidity of precocious puberty has increased gradually these decades,and the menarche age of girls is becoming earlier and earlier.Adolescence is a transitional period in which reproductive capacity gradually matures,accompanied by significant changes in body,behavior,and psychology.Precocious puberty may lead to a short final adult height of children and have adverse effects on children’s psychology and behavior.What’s more,it may increase the risk of cardiovascular disease and metabolic disease after adulthood.The process from puberty initiation to sexual maturity is a highly energy-consuming process,so good nutritional status and sufficient energy reserve are the necessary conditions for puberty initiation.Ghrelin is a peptide hormone secreted in stomach.It regulates energy metabolism,reduces fat utilization,increases fat accumulation and leads to body weight gain,and even may participate in the control of puberty.However,the role and mechanism of ghrelin in female precocious puberty are still unclear.There are two forms of ghrelin exiting in vivo:acylated ghrelin(AG)and unacylated ghrelin(UAG).AG is considered to be the main form of ghrelin to play its physiological role in vivo,but more and more researchers have confirmed that UAG can also perform the physiological role of ghrelin in vivo.In this study,we followed up with the girls with precocious puberty,and analyzed their serum levels of total ghrelin(TG),AG and UAG.Then the role and relationship between TG,AG,and UAG,and precocious puberty were discussed in those girls.Subsequently,to verify the role of TG,AG and UAG in precocious puberty,we built a precocious mice model with a high fat diet.After that,we studied the changes in serum TG,AG,and UAG levels in precocious mice.Finally,to explore the central mechanism of ghrelin in the onset of puberty of female precocious puberty,the whole cell patch clamp technique was used to record the electrophysiological properties of Gonadotropin-releasing hormone(Gn RH)neurons and the changes of them before and after ghrelin administration.Part Ⅰ The Relationship between Ghrelin and the Onset of Puberty in Girls with Precocious PubertyObjective:The aim of this study was to investigate the changes in TG,AG,and UAG levels in girls with precocious puberty.And then analyzed and explored the relationship between serum TG,AG and UAG levels and incidence of precocious puberty in girls.Methods:22 girls diagnosed with idiopathic central precocious puberty(ICPP)were collected as the ICPP group and 28 healthy girls at the same age were recruited as the control group.We collected age,bone age,height,weight,and some other information of the girls recruited,and calculated body mass index(BMI).The levels of TG and AG were detected through ELISA.The level of UAG was the subtractive value of TG and AG.Besides,ICPP girls needed Gn RH stimulating tests additionally.Results:(1)There were no differences between the age of the ICPP group and the control group,which were(8.16±0.17)years and(7.73±0.22)years respectively.The weight of the ICPP girls was significantly higher than that in the control group[(30.66±1.28)kg vs.(24.16±0.98)kg],and so was BMI[16.38(18.28-14.94)kg/m~2 vs.15.02(16.00-13.52)kg/m~2],height[(135.20±1.57)cm vs.(126.10±0.76)cm]and bone age[(10.53±0.27)years vs.(7.60±0.25)years].On the contrary,the AG level of girls in the ICPP group(920.76±20.11)pg/m L was lower than that in the control group(993.73±11.77)pg/m L.All the differences above were statistically significant(P<0.05).Results of ICPP girls’Gn RH stimulating test were as follows:peak luteinizing hormone(LH)was(20.82±14.58)m IU/L,peak follicle-stimulating hormone was(14.01±4.49)m IU/L,and peak LH/peak FSH ratio was 1.44±0.77.The levels of TG and UAG between the two groups existed no statistical significance.(2)ROC curve showed that 1/AG and body weight had higher sensitivity and specificity for the female ICPP diagnosis.(3)Pearson correlation analysis found that AG was negatively correlated with bone age,height,and weight(P<0.05).(4)Single-factor logistic regression analysis found that OR odds of AG was 0.988,95%confidence intervals were(0.979,0.996)(P<0.05),which was not interfered by age and obesity;OR odds of weight was 1.234,95%confidence intervals were(1.080,1.410)(P<0.05).Conclusions:(1)Compared with the control group,serum AG level of the ICPP group was significantly lower;AG was negatively correlated with weight,height and bone age;(2)AG helps to identify ICPP in girls,and AG may reduces the risk of precocious puberty in girls;(3)Weight helps to identify ICPP in girls,and weight may increase the risk of precocious puberty in girls.In conclusion,lower AG level and obese may be the indicators of female precocious puberty.Part Ⅱ The role and mechanism of Ghrelin in puberty onset of female precocious miceObjective: This study was designed to analyze the levels of TG,AG,and UAG levels in female precocious mice,to analyze the effect of ghrelin on the electrophys-iological characteristics of Gn RH neurons,and explore the role and mechanism of ghrelin in female precocious puberty.Methods: Animals were C57BL/6J Gn RH-GFP mice.We divided them into two groups: the control group(standard diet group,Ctrl)and the experimental group(high-fat diet group,HFD).The model of female mice with precocious puberty was established by using a high-fat diet.The mother mice were given a standard diet or a high-fat diet on the day of delivery.On the 21 st day(PN 21d),the cages were divided and the time of vaginal opening was observed for the first time,and the diet before cage division was continued.The female mice fed with a standard diet were enrolled in the Ctrl group.The female mice fed with the high-fat diet,whose time of vaginal opening was earlier than that of the Ctrl group,were enrolled in the HFD group.On one hand,observe and analyze the differences and relationship between sex hormone levels(E2,LH,FSH)and TG,AG,and UAG of the mice.On the other hand,the electrophysiological characteristics of Gn RH neurons in the two groups were compared before and after 100 nmol/Lghrelin perfusion.Results:(1)From the 21 st day on,the weight of the HFD group was significantly higher than that in the Ctrl group(P < 0.001);The vaginal open time of HFD group was 30.00(30.00-29.75)d,which was significantly earlier than Ctrl group 40.00(41.50-35.00)d(P < 0.0001);therefore,the point-in-time to explore the mechanism of why high-fat diet leads to the earlier onset of puberty was set at PN 30 d.(2)On the 30 th day,the LH level in the HFD group was significantly higher than that in the Ctrl group [1.25(3.37-0.56)m IU/m L vs.0.38(0.65-0.14)m IU/m L];but the TG and AG level were lower than those in Ctrl group [(675.23±8.95)pg/m L vs.(752.80±14.51)pg/m L,336.20(339.90-333.30)pg/m L vs.369.10(388.10-340.30)pg/m L,respectly],all the differences were statistically significant(P < 0.05);(3)ROC curve analysis showed that 1/AG had higher sensitivity and specificity for ICPP diagnosis with female mice;(4)On the 30 th day,when mice of HFD group had reached puberty,the action potential(AP)half-width AP amplitude and AP firing frequency of Gn RH neurons decreased and RMP increased,compared with Ctrl group;(5)Ghrelin inhibited the excitability of Gn RH neurons via increasing AHP,decreasing Rin and action potential(AP)firing frequency.After ghrelin perfusion,the absolute decreased values of AP firing frequency and the AP firing frequency decreased rate for every minute in the HFD group were more significant than the Ctrl group.Conclusions:(1)Fed with high-fat diet in the early life stage can lead to early puberty in female mice;(2)Low TG and AG levels may be one of the reasons for the early puberty of female rats;(3)AG is helpful to identify precocious puberty in female rats;(4)Ghrelin could lower the excitabilities of Gn RH neurons,and the inhibitory effect on Gn RH neurons in HFD group was stronger than that in Ctrl group;In a word,both girls and female mice that with precocious puberty,whose level of ghrelin was lower than normal condition,so it may be one of the mechanisms of female precocious puberty by reducing the inhibitory effect on the electrophysi-ological activities of Gn RH neurons.